In article <iain.shaw-1604970938170001 at pc0776.irad.bbsrc.ac.uk>,
iain.shaw at bbsrc.ac.uk (Iain Shaw) wrote:
> Dear All,
>> I am currently cloning PCR reagents into invitrogen TA kit, quite
> succesfully but at quite a high cost. What I would like to do is make my
> own by cutting pBluescript and add a single T overhang. I have a
> T-tailing kit from Boehringer (DNA Tailing Kit), but you do not appear to
> be able to limit the amount of Ts that are added. Does anyone know a way
> of adding a single T this way, or has anyone any suggestions on how else
> to do this.
>
iain,
here's what we did to make our own in house TA vector.
we made two complementary oligos that, when annealled had sticky EcoRI
ends and contained 2 XcmI sites in the middle. XcmI has a recognition
sequence that looks like this....
5' 3'
CCANNNNN NNNNTGG
GGTNNNN NNNNNACC
we made the oligos so that the first of the 2 XcmI sites had a T on the
top 3' overhang and the second one had a T on the bottom 3' overhang. we
then subcloned the annealled oligo into EcoRI cut Bluescript and when we
want to make some TA vector we just cut with XcmI, gel purify (some people
in the lab CIP the vector and others do not with similar results) and use
it in a ligation just like the Invitrogen version.
one caveat is that it is not very stable at either 4oC or -20oC. in our
hands it needs to be re-made after a week at most. i have not tried
drying it down (like invitrogen used to do) or storing it at -70oC, but
those are on my list of things to do before i leave here in a few months.
if you have any more questions, please feel free to ask and i'll do what i
can to answer them.
eric
--
Eric C. Anderson
Memorial Sloan-Kettering Cancer Center
Sloan-Kettering Institute
1275 York Ave. Box 470
New York, NY 10021
(212) 639-2977
e-anderson at ski.mskcc.org
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